Transgenic technology to convert animals into “bioreactor” for the production of specific proteins or other substances of pharmaceutical interest (Gordon et al., 1987, Biotechnology 5: 1183-1187; Wilmut et al., 1990, Theriogenology 33: 113-123) offers significant advantages over more conventional methods of protein production by gene expression.
Recombinant nucleic acid molecules have been engineered so that an expressed heterologous protein may be joined to a protein or peptide that allows secretion of the transgenic expression product into milk or urine, from which the protein may then be recovered. These procedures may require lactating animals, with the attendant costs of maintaining individual animals or herds of large species, such as cows, sheep, or goats.
Historically, transgenic animals have been produced almost exclusively by microinjection of the fertilized egg. The pronuclei of fertilized eggs are microinjected in vitro with foreign, i.e., xenogeneic or allogeneic, heterologous DNA or hybrid DNA molecules. The microinjected fertilized eggs are then transferred to the genital tract of a pseudopregnant female (e.g., Krimpenfort et al., U.S. Pat. No. 5,175,384).
One system that holds potential is the avian reproductive system. The production of an avian egg begins with formation of a large yolk in the ovary of the hen. The unfertilized oocyte or ovum is positioned on top of the yolk sac. After ovulation, the ovum passes into the infundibulum of the oviduct where it is fertilized if sperm are present, and then moves into the magnum of the oviduct, which is lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin, into the lumen of the magnum where they are deposited onto the avian embryo and yolk.
The hen oviduct offers outstanding potential as a protein bioreactor because of the high levels of protein production, the promise of proper folding and post-translation modification of the target protein, the ease of product recovery, and the shorter developmental period of chickens compared to other potential animal species. The chicken ovalbumin gene is highly expressed in the tubular glands of the mature hen oviduct and is therefore a suitable candidate for an efficient promoter for heterologous protein production in transgenic birds. Efforts have been made to create transgenic chickens expressing heterologous proteins in the oviduct by means of microinjection of DNA (PCT Publication WO 97/47739).
Gene expression must be considered not only from the perspective of cis-regulatory elements associated with a gene, and their interactions with trans-acting elements, but also with regard to the genetic environment in which they are located. Chromosomal positioning effects result in variations in levels of transgene expression associated with different locations of the transgene within the recipient genome. An important factor governing the level of transgene expression is the chromatin structure around a transgene, and how it cooperates with the cis-regulatory elements. While the deletion of a cis-regulatory element from a transgenic lysozyme locus can be sufficient to reduce or eliminate positional independence of the level of gene expression, there is also evidence that positional independence conferred on a transgene requires the cotransfer of many kilobases of DNA other than just the protein encoding region and the immediate cis-transcriptional regulatory elements. Scattered throughout the chicken genome, including the chicken ovalbumin locus, are short sequences that resemble features of Long Terminal Repeats (LTRs) of retrovirus. The function of these elements is unclear but most likely may help define the DNAse hypersensitive (DHS) regions of a gene locus (Stein et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 6485-6489). Thus, flanking various avian genes are matrix attachment regions (5′ and 3′ MARs), alternatively referred to as “scaffold attachment regions” or SARs. The outer boundaries of the chicken lysozyme locus, for example, have been defined by the MARs (Phi-Van et al., 1988, E.M.B.O.J. 7: 655-664; Phi-Van & Stratling., 1996, Biochem. 35: 10735-10742). Deletion of a 1.32 kb or a 1.45 kb region, each comprising half of a 5′ MAR, reduces positional variation in the level of transgene expression (Phi-Van & Stratling, supra).
The 5′ matrix attachment region (5′ MAR), located about −11.7 kb upstream of the chicken lysozyme transcription start site, can increase the level of gene expression by limiting the chromosomal positional effects exerted against a transgene (Phi-Van et al., 1988, supra). At least one other MAR is located 3′ downstream of the protein encoding region. Although MAR nucleic acid sequences are conserved, little cross-hybridization is seen, indicating significant overall sequence variation. However, MARs of different species can interact with the nucleomatrices of heterologous species, to the extent, for example, that the chicken lysozyme MAR can associate with the plant tobacco nucleomatrix as well as that of the chicken oviduct cells (Mlynarona et al., 1994, Cell 6: 417-426; von Kries et al., 1990, Nucleic Acids Res. 18: 3881-3885). The lysozyme promoter region of chicken is also active when transfected into mouse fibroblast cells and linked to a reporter gene such as the bacterial chloramphenicol acetyltransferase gene. In each case, the presence of a 5′ MAR element increased positional independency of the level of transcription (Stief et al., 1989, Nature 341: 343-345; Sippel et al., pgs. 257-265 in Houdeline L. M. (ed), “Transgenic Animals: Generation and Use”).
The ability to direct the insertion of a transgene into a site in the genome of an animal where the positional effect is limited offers predictability of results during the development of a desired transgenic animal, and increased yields of the expressed product. Sippel and Steif disclose, in U.S. Pat. No. 5,731,178, methods to increase the expression of genes introduced into eukaryotic cells by flanking a transcription unit with scaffold attachment elements, in particular the 5′ MAR isolated from the chicken lysozyme gene. The transcription unit disclosed by Sippel and Steif was an artificial construct that combined only the −6.1 kb enhancer element and the proximal promoter element (base position −579 to +15) from the lysozyme gene. Other promoter associated elements were not included.
Although individual cis-transcriptional regulatory elements associated with the chicken ovalbumin gene have been isolated and sequenced, together with short regions of flanking DNA, the entire nucleic acid sequence comprising the 5′ upstream region of the ovalbumin gene has not been determined and has not been employed as a functional promoter to allow expression of a heterologous transgene.
What are still needed, however, are efficient transcription promoters that allow expression of transgenes in avian cells but with reduced positional variation.
What is also still needed is a gene expression promoter cassette that will allow expression of a transgene in the oviduct cells of an avian and efficient gene expression regardless of the chromosomal location of the expression system.